Latin American applied research

versión impresa ISSN 0327-0793

Resumen

Many publications describe methods for peroxidase purification from plant material. When the goal was to obtain a high purity enzyme every purification method included an affinity chromatography step using Concanavalin A as the ligand. However, the adsorption step was carried out under quite different conditions with regard to pH, ionic strength and metal cation content in the binding buffer, thus leading to a rather confuse situation. To establish the best conditions for purification of peroxidases from horseradish root (HRP) and soybean hull (SBP), equilibrium adsorption isotherms were fitted to the Langmuir-type model, where ionic strength, pH and cation concentration were chosen as the variables. For SBP, our results showed that Kd rounded 10-6 M in all cases (pH 5.0 - 7.0, 1 and 5 mM Mn2+ / Ca2+, 0 - 0.75 M NaCl). For HRP, Kd ranged between 10-5 M and 10-6 M depending on the parameters. Under optimised binding conditions, 84.3% SBP was recovered after elution carried out with 0.74 M a -methyl-D-mannopyranoside and 0.37 M NaCl. For HRP, the recovery was lower (75%) and 0.36M a -methyl-D-mannopyranoside was necessary for the elution step.